2014 Predavanje Broj 3 Manipulisanje genima

8/11/2019 2014 Predavanje Broj 3 Manipulisanje genima

1/56

Seminarski rad 1: Opisati postupak kloniranja gena organizma

iji genom jo uvek nije sekvenciran

GRUPA 1: studenti sa parnim rednim brojevima na listi

Veliina nepoznatog gena oko 40 Kb

Pojedini delovi gena koji kodiraju odreene proteinske domene pokazuju visokstepen homologije na nukleotidnom nivou sa odgovarajuim regionima ortolognih

gena ije su sekvence poznate i deponovane

Navesti ta je potrebno za postupak kloniranja

Dati detaljan prikaz eksperimenta kloniranja

GRUPA 2 : studenti sa neparnim rednim brojevimana listi

Veliina nepoznatog gena oko 5 kb

Gen pokazuje izuzetno visok stepen homologije na nukleotidnom nivou saortolognim genima ije su sekvence poznate i deponovane

Navesti ta je potrebno za postupak kloniranja

Dati detaljan prikaz eksperimenta kloniranja

Seminarski rad otprintovan na najvie 2 straneformata A4 sa imenomstudenta, brojem indeksa i potpisom dostaviti na poetku predavanjakoje e

biti odrano 11.04.2013.g.


2/56

Organism GenomeSize(Mb)

Gene Number

Hepatitis D virus 0.0017 1

Hepatitis B virus 0.0032 4

HIV-1 0.0092 9

Bacteriophage l 0.0485 80

Escherichia coli 4.6392 4400

S. cerevisiae(yeast) 12.155 6300

C. elegans (nematode) 97 19000

D. melanogaster(fruit fly) 137 13600Mus musculus(mouse) 3000 20000-30000

Homo sapiens(human) 3000 20000-30000

1 Mb = 1 million base pairs (for double-stranded DNA or RNA)

The genomes of prominent organisms
http://www.ncbi.nlm.nih.gov/genomes/framik.cgi?db=genome&gi=10565http://www.web-books.com/MoBio/Free/Ch3H1.htmhttp://www.web-books.com/MoBio/Free/Ch3H2.htmhttp://www.ncbi.nlm.nih.gov/genomes/framik.cgi?db=genome&gi=10119http://www.web-books.com/MoBio/Free/Ch3H3.htmhttp://www.web-books.com/MoBio/Free/Ch3H5.htmhttp://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?chr=celegans.infhttp://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=7227http://www.ncbi.nlm.nih.gov/genome/guide/mouse/http://www.ncbi.nlm.nih.gov/genome/guide/humanhttp://www.ncbi.nlm.nih.gov/genome/guide/humanhttp://www.ncbi.nlm.nih.gov/genome/guide/mouse/http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=7227http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?chr=celegans.infhttp://www.web-books.com/MoBio/Free/Ch3H5.htmhttp://www.web-books.com/MoBio/Free/Ch3H3.htmhttp://www.ncbi.nlm.nih.gov/genomes/framik.cgi?db=genome&gi=10119http://www.web-books.com/MoBio/Free/Ch3H2.htmhttp://www.web-books.com/MoBio/Free/Ch3H2.htmhttp://www.web-books.com/MoBio/Free/Ch3H2.htmhttp://www.web-books.com/MoBio/Free/Ch3H1.htmhttp://www.ncbi.nlm.nih.gov/genomes/framik.cgi?db=genome&gi=10565


3/56

E.coligenome

CIRCULAR MAP

Genetic map: linkagemap of known genes or

functional sites Often given in minutes(the time which it takesto each malechromosome to move

into female cell) Physical: restriction

sites of mapped genes


4/56

Organisation ofE.coligenome A prokaryotic genome has to squise into

relatively tiny space with the help of DNAbinding proteins that package the genome in

an organised fashion

The circular genome is supercoiled: in atypical prokaryote the genome is a singlecircular DNA molecule is organized in

NUCLEOID

The protein component of nucleoid includeDNA gyraseand DNA topoisomerase I- the

two enzymes that are primarly responsible formaintaining the supercoiled state

A set of at least four additional proteinsarebeleived to have a more specific roles in

packaging of bacterial DNA

The circularE. colichromosome has acircumference of 1.6 mm(E.colicell has

dimension 1.0 x 2.0 mm)

Remove of a few turns

of the doublehelix (underwinding)

results

in negative supercoiling

NUCLEOID


5/56

Bacteria genome is a nucleoid

Although bacteria do not display structure with the distinctmorphological features of the eucaryotic chromosomes, bacteria

genomes are organized into definite bodies-NUCLEOID.

Protein core

Between 50 and

100 supercoiled

loops of DNA

radiate from the

central protein

core


6/56


7/56


8/56


9/56


10/56


11/56

Structures of newRhselements

Unique DNA sequences from theE. coliK-12 chromosomal framework aredesignated ORFs f256, o205, and o77 (2). TheRhselements are aligned so that thestart codon of the core ORF is base 1.RhsEandRhsHof ECOR-45 are linked intandem (indicated by the diagonal dashed line). The core of ECOR-45RhsGcontains a 587-bp deletion, indicated by .

Wang etal., JBC

Journal of Bacteriology,

1998, 180, 4102-4110.


12/56


13/56

Transposable Elements in Prokaryotes

Insertion Sequences (IS)

Noncomposite transposons

Composite Transposons


14/56


Simplest transposable elements: Segments of DNA in bacteriathat can move from one position to another.

Normal constituents of bacterial chromosome and plasmids.

Cause increase in the size of the genome

IS elements only contain genes required to mobilize theelement and insert the element at a new location.

When IS elements transpose, promoters within IS elementsthemselves may alter expression of nearby genes.


15/56

Insertion Sequences: IS

Insertion elements are mobile genetic elementsthat occasionally insert intochromosomal sequences, often disrupting gene.

IS are a special class of transposable elements found in prokaryotes(studied extensively inEscherichia coli K12)

They usually range in size from 1 to 2 kilobasepairs(kb) and containperfect or nearly perfect inverted terminal repeatsequences. These terminalrepeats likely are recognition sites for an enzyme responsible for the

insertion.

Mobility of the element depends only on the element itself; it is an

autonomous element. Thus, it must carry the coding ability for thetransposase recognizing the inverted terminal repeats.


16/56


The terminal sequences flank a unique central sequence with at least one long openreading framecoding, presumably, for the transposase protein: All IS elements are

made up of transposae gene flanked by inverted repeats(IRs).

IS1first identified inE. colis glactose operon is 768 bp long and is present with 4-19 copies in theE. colichromosome

Ends of all known IS elements show inverted terminal repeats (ITRs)


17/56

Mechanizm of IS insertion Staggered cut made at target site

IS element inserted, joined to singlestranded ends: DNA Pol and DNAligase fills in gaps.

Produced target site duplicationflanking the IS element.

The direct repeats externallyflanking the inverted repeats are notpart of the insertion sequence.Instead, they are chromosomalsequences that become duplicatedupon insertion, with one copy ateach end; this is called target-site

duplication. Orientation of IS Elements: IS

elements can insert in anyorientation.


18/56

Composite Transposons

Created when two IS elements insert near each other. The region betweenthem can then be transposed by joint action of flanking IS elements (i.e.

two IS elements "capture" a DNA sequence and endow it with the ability totranspose).

In many cases, these "captured" sequences are antibiotic resistance genes.These transposons can jump onto conjugation plasmids and be transferred

from one strain or even one species to another. This is of great medicalsignificance.

R-factorsare multidrug-resistance plasmidscontaining multipletransposons bearing different antibiotic resistance genes.

Compositetransposons:

Have two ISs at

their ends, theDNA between thetwo ISs canencode resistancegenes or virulencefactors. In manycases only one of

the twotransposases isfunctional.


19/56

Noncomposite transposons

Noncomposite transposons combine the qualities of both IS elements andcomposite transposons.

Like IS elements, they have single IR sequences at each end.

Like composite transposons, they carry selectable marker genes.

Consists of a transposase gene, plus other genes, flanked by invertedrepeat (IR) sequences

IR sequences can form "stem-loop" structures when DNA containing transposon is

denatured and ssDNA allowed to reanneal.


20/56

Evolutionary role of insertion sequences

Selfish or parasitic DNAsequences which are maintained in the population, even inthe face of adverse natural selection, as a result of their ability to transpose and

become horizontally transmitted among strains by means of hitchhiking inplasmids.

Insertion sequences also play an important role in the evolutionof transposons and

plasmids: One evolutionary implication of insertion sequences derives from their mutagenicactivity in causing insertion mutations.

A pair of insertion sequences flanking a central sequence can transpose as a unit,and such composite transposons containing antibiotic-resistance genes, for

example,Tn5, Tn9 and TnlO, are well documented

Insertion sequences and transposons can also transpose into plasmids and remoldtheir structure, and in general change the genetic capabilities of plasmids

The distribution of numbers of insertion sequences in the genome is also of someinterest in understanding the population dynamics of the elements.


21/56


22/56

Streptomycetes

Comprises a large group of filamentous soil gram-

positive bacteria

The most important group of industrial

microorganisms: producing approximately two-thirdsof all known varieties of antibiotics

Produces many other useful secondary metabolites

and extra cellular enzymes


23/56

Phenomenon of genetic instability

Instability of certain genetic traits: mutations frequently exhibit

pleiotropy- several traits being altered at the same time Molecular studies have shown that trait losses are due to largedeletions (ten to thousands of KB long) in the chromosomal DNA

These large chromosomal DNA (in average 8 MB in size) haveterminal inverted repeats

Deletions are frequently accompanied by tandem amplifications ofup to several hundred copies of specific DNA sequences termedAmplifiable Units of DNA (AUD)

This phenomenon is explained by fact that chromosomesof manyStreptomycetes species are linear DNA molecules

Proteins are covalently attached to the 5-terminal ends of

chromosomal DNAs which presumably act as the primers ofreplication

Unstable regions are at the both termini of the chromosome

Essentially all deletions involve one, or both, telomeres


24/56

Complete genome sequence of the model actinomycete

Streptomyces coelicolorA3(2), 2002, Nature 417, 141 -147 S coelicoloris a soil bacterium that has

many different metabolic processes andbiotransformations that allow it to liveunder a wide range of conditions in thesoil and use a wide range of metabolic

pathways to, for example, degrade

insoluble remains of other organisms. S coelicoloris unusual, in that it is a

multicellular bacterium which forms intodifferent sorts of 'tissues'. The complexityof its metabolic pathways give rise to twothirds of modern antibiotics, as well as

anti-tumour and immuno-suppressantagents.

All this complexity arises from the longestknown eubacterial genome (8.7million

base pairs and 7,825 genes; about onequarter the number of human genes).


25/56


26/56

Yeast Genome


27/56

Yeast Genome:Saccharomyces cerevisiae

The entire genome sequence was released in April, 1996 and was the first

eukaryotic genome to be sequenced. The haploid genome contains 16 chromosomesranging in size from 220 to 1500 kb

Analysis of the genome reveals 6,183 potential ORFs

Compared to the genomes of other organisms, the yeast genes are very efficientlyspacedon the 16 chromosomes with a density of 1 gene/2 kb of DNA.

Most yeast genes do not contain introns

There is little 'junk' DNAin the intergenic regions commonly seen in other eukaryotes. A total genome size of approximately 13,000kb: Chromosomes 85%of the total yeast DNA

The 2mm-plasmid accounts for about 5%,

Mitochondrial DNA 10%

The 6.3 kb 2mm-plasmidplasmid has no known function in yeast other than toreplicate, but it is not deleterious to the cell. Each cell contains 60-100 copiesof

this plasmid. Researchers have taken advantage of this naturally occurring highcopy number plasmid to create yeast plasmid vectorscontaining the 2mm-plasmid

origin of replication.


28/56

Life with 6000 genesScience,1996, Vol. 274, p546

Reports on the complete sequencing of the genome of theyeast Saccharomyces cerevisiae.

The sequence of 12,068 kilobases defining:

6183 potential ORFsof at least 100 amino acids in length. However,

there are likely additional ORFs smaller than 100 amino acids and notall of the 6,183 potential ORFs are protein encoding genes.

5885potential protein-encoding genes

140genes specifying ribosomal RNA;

40genes for small nuclear RNA molecules;

275transfer RNA genes Providing of information about the higher order organization

of yeast's 16 chromosomes and allowance of some insight intotheir evolutionary history


29/56

The total number of real protein-coding genes

It has been proposed a revised yeast gene catalog consisting of 5538 ORFs 100 amino acids.This reflects the proposed elimination of 503 ORFs.

Only two-thirds of these have been experimentally validated (known), and the remaining~2000 ORFs are currently annotated as hypothetical. The total number of real protein-codinggenes has been a subject of considerable debate, with estimates ranging from 4,800 to 6,400

genes.


30/56

Saccharomyces cerevisiaecomplete

genome

The size of DNA in each chromosome of the yeast S. cerevisiae.


31/56

R t t


32/56

The elements are some 6 kb flanked by long terminal repeats (LTRs; some 300 bp in length),which are named delta (for Ty1/2), sigma (for Ty3) and tau (for Ty4).

Ty elements resemble retroviral genomes in yet other aspects: there are two overlapping (in a

+1 mode) open reading frames. ORF TyAencodes the gag(envelope) protein of the virus-like particles; TyBprovides a polycistronic message the product of which is processed by the endogenous Asp-type

protease to yield a Ty integraseand a retro transcriptase.

Though the elements share a relatively high degree of homology,Ty1/2/4 belong to the 'copia'class of retroelements, while Ty3 is a member of the 'gipsy' family.

The majority of the Ty elements were found to transpose upstream of tRNAgenes, in asequence non-specific manner. These sites appear to represent less harmful targetsthan genes

or other intergenic regions.

Retrotransposons

in yeast

-Ty1- Ty5.-Ty1 through Ty4 havebeen identified to

retrotranspose via an

RNA intermediate

-Ty5 does appear to be

no longercapable of transposition


33/56

The genome sequence of Schizosaccharomyces pombe

Nature. 2002 Feb 21;415(6874):871-80.

The genome of fission yeast (Schizosaccharomyces pombe) contains thesmallest number of protein-coding genes yet recorded for a eukaryote:4,824

The centromeres are between 35 and 110 kilobases (kb) and contain relatedrepeats including a highly conserved 1.8-kb element.

Regions upstream of genes arelongerthan in budding yeast

(Saccharomyces cerevisiae), possibly reflecting more-extended controlregions.

Some 43% of the genes contain introns.

50 genes have significant similarity with human disease genes; halfof theseare cancer related.

Highly conserved genes important for eukaryotic cell organization

including those required for the cytoskeleton, compartmentation, cell-cyclecontrol, proteolysis, protein phosphorylation and RNA splicing areidentified. These genes may have originated with the appearance of

eukaryotic life.

Few conserved genes that are important for multicellular organizationwereidentified, suggesting that the transition from prokaryotes to eukaryotes

required more new genes than did the transition from unicellular tomulticellular organization.


34/56


35/56

Genetic mapping

A pair of homologous chromosomes can exchange parts bycrossing-over

Recombination produces genotypes with new combinationsof

parental alleles

Two genes close together on the same chromosome pair do notassort independently at meiosis

Gene loci on chromosome can be mapped by measuring thefrequencies of recombinantsproduced by crossing-over


36/56

Meiotic recombination

Process that generates a haploid product with

genotype that differs from both haploid genotypes

that constituted the meiotic diploid cell

The product of meiosis generated by recombination is

called RECOMBINANT


37/56

Haplotype

Recombination will rarely separate loci which lie close

together on a chromosome

A set of alleles on the small chromosomal segment tend to be

transmitted as a block through a pedigree

Such block of alleles is known as HAPLOTYPE

Haplotypes mark recognizable chromosomal segments which

can be traced through pedigrees and through population


38/56

Genetic distance

The recombination fraction is a measure of the

distance between two loci

The recombination fraction define genetic distance

Two loci which show 1% recombinationare defined

as being 1 CENTIMORGANapart on a genetic map


39/56

a)

b)

a

b

A B

A B

a b

a B

A b

a b

A B

a b

A B

a b

a B

A b

Heterozigotni roditelj

Produkti mejotike

rekombinacijeDobijena

frekvenca

45%

45%

5%

5%

90% roditeljski

10% rekombinant

Heterozigotni roditelj

Produkti mejotike

rekombinacije Oekivana

frekvenca

25%

25%

25%

25%

50% roditeljski

50% ne-roditeljskiNezavisna segregacija


40/56

Genetic markers

Mendelian characters which are sufficiently polymorphic to

give a reasonable chance that randomly selected person will be

heterozygous

Highly polymorphic and informativeRandomly distributed

Easy to score

High PIC valuesPIC (polymorphism information content) a measure

of the informativness of genetic marker


41/56


42/56

STS(sequence tagged site): any piece of DNA

whose sequence is known and for which aspecific PCR assay has been designed

EST(expressed tagged site): a short sequence

of cDNA for which a PCR assay is available


43/56

RFLP

Restriction Fragment Length Polymorphism

A polymorphism due to difference in size ofallelic restriction fragments as a result of

restriction site polymorphism


44/56

Effects at the protein levelEffects at the DNA level

Normal red blood cells (top)

and sickle cells (bottom)

There are effects at the cellular level.

When red blood cells carrying mutant hemoglobin are

deprived of oxygen, they become sickle-shaped instead of the

usual round shape (see picture). This shape can sometimes

interrupt blood flow.There are negative effects at the whole organism level.

Under conditions such as high elevation and intense exercise, a carrier of

the sickle cell allele may occasionally show symptoms such as pain and

fatigue.

There are positive effects at the whole organism level.

Carriers of the sickle cell allele are resistant to malaria, because theparasites that cause this disease are killed inside sickle-shaped blood cells.

Sickle Cell Anemia


45/56

RFLP and sicklecell mutation

CCTTAGG

GGAATCC

CCTGAGG

GGACTCC

CCTTAGG

GGAATCC

CCTTAGG

GGAATCC

CCTGTGG

GGAAACC

CCTTAGG

GGAATCC

0.2 KB 1.1 KB

1.3 KB

Mst I I Mst I I Mst I I

Mst I IMst I I

ba

bs


46/56

RFLP and sickle cell mutation


47/56

Vezanost markera

Kada se ispituje odreeno svojstvo (osobina, oboljenje) prvi

korak je odreivanje na kom hromozomu se nalazi traeni

genski lokus

To se radi indirektno traenjem neke druge genetike

karakteristike (odnosno genetikog markera) koji se nasleuje

u korelaciji sa bolesnim alelom

Vezanost je korelacija u nasleivanju nekog RFLP alela (ili

nekog drugog genetikog markera) i bolesnog alela

Li k A l i li i


48/56

Linkage Analyis: analiza vezanosti

markera

Tendencija da se markeri na specifinom lokusu

nasleuju zajedno kao posledica njihove fizikevezanosti na pojedinanim hromozomima


49/56

A probe P detects two DNA

morphs when DNA is cut

by a certain restriction

enzyme (RE).

The pedigree of analysis of

dominant disease phenotype

D shows:

-Linkage of the D locus tothe RFLP locus in childs 2,

3, 6 and 7

-Absence of linkage in child

8 ?????

The detection and inheritance of RFLP

i


50/56

The detection

and

inheritance of

RFLP

The pedigree of

dominant diseasephenotype D shows

linkage of the D locus to

the RFLP locus.

Only child 8is recombinant


51/56

Mapping using microsatellite repeats as

molecular markers

Disease allele P is probably linked to M


52/56

Mapping using DNA Fingerprint bands as molecular marker

DNA fingerprint

The specific patternof DNA fragments

formed when DNA

is cut with

restriction enzymes,

separate and

hybridize

Alele P possible

linked to C or I

A

BCDE

F

G

I

H

F and H: Always inherited together- linked?

A and B: in progeny always either A or B- allelic?

A and D: Four combinations; A and D, A, D or neither- unlinked?F, H and E: Always either F or H or E- closely linked in trans?


53/56

DNA profiling

In violent crimes such a murder or rape andin paternity (or grand-paternity) cases.

Fortunately DNA is quite stable and resistantto degradation.

With the latest procedures availablesufficient DNA may be obtained from just asingle hair follicle.

Autoradiograph from an actual rape caseshowing the DNA profiles for one VNTRlocus.

The lanes marked "M" show a "ladder" ofDNA fragments of known sizes. These areloaded onto the gel to provide an internal

ruler--allowing the sizes of the VNTR allelesto be estimated more accurately.

As can be seen there is a match of the DNAprofile of defendant 1 and the forensicsample.


54/56

LOD SCORE

Mera verovatnoe o genetikoj vezanosti

izmeu lokusa

Lod score > +3 dokaz vezanost

Lod score < -2 dokaz odsustva vezanosti


55/56


56/56

Documents

2014 Predavanje Broj 3 Manipulisanje genima